Interferons and other immunomodulators (i.e. BCG) are used as front-line therapy in patients with bladder ancer, but their mechanisms of action remain unclear. Interferons have potent anti-angiogenic effects that appear to contribute to their anti-tumor activities. In addition, in studies performed by our laboratories during the first cycle of SPORE funding, we demonstrated that interferon-a (IFNa) induced apoptosis in a subset (6/20) of human bladder cancer cell lines and that IFNcc-induced expression of tumor necrosis factor apoptosis-inducing ligand (TRAIL) was involved. In parallel studies we demonstrated that some bladder cancer cells are IFN-resistant because they are resistant to TRAIL. Importantly, we showed that resistance to IFN or TRAIL could be reversed by incubating them with the proteasome inhibitor, bortezomib (PS-341, Velcade) or the histone deaceylase inhibitor, SANA, and our preliminary data strongly suggest that they do so by promoting the p53-independent accumulation of the cyclin-dependent kinase inhibitor, p21 Waf-1/Cip-1. The overall goal of the studies proposed in this competing renewal is to use this information to design an effective, death receptor-based therapeutic strategy for the treatment of urothelial cancer. To this end, we propose the following 3 Specific Aims. (1) Define the role of p21 in bortezomib- or SAHA-mediated sensitization of urothelial carcinoma cells to IFN or TRAIL. (2) Evaluate the efficacy and toxicity of TRAIL- or IFN-based combination therapies in orthotopic bladder tumors in vivo. (3) Develop methods to measure pharmacodynamic markers of biological response to bortezomib-based therapy in patients. These experiments will complement parallel studies being performed in Projects 4 and 5, where alternative strategies to enhance TRAIL sensitivity and/or bypass the TRAIL pathway altogether are being explored. The most important distinction between this project and previous work with biological agents is that it appears that cytostatic agents promote TRAIL-induced apoptosis in tumor cells, whereas they can undermine the effects of many (if not most) conventional chemotherapeutic agents. All of the compounds being studied are either already FDA-approved for the treatment of cancer or are in the process of being evaluated in Phase l-lll clinical trials. Therefore, an important feature of this project is that we will open a clinical trial of our most promising combination by the 4th year of SPORE funding.